人胎儿血造血干／祖细胞的生物学特性及其移植实验

背景：造血干细胞来源目前包括骨髓干细胞、外周血干细胞和脐带血干细胞，寻找新的干细胞来源以满足临床移植需要一直是人们的希望。从妊娠第5周起，肝脏中发育成完善的血窦系统，此后造血干细胞就可以随血液流动迁移。 目的：观察人胎儿血造血干／祖细胞的生物学特性并对其进行非肥胖糖尿病／重症联合免疫缺陷小鼠移植。 设计：对照实验。 单位：广西医科大学第一附属医院血液科。对象：①细胞来源：21例胎儿血标本取自胎龄为18-29周[（24．2&;#177;3．2）周1死亡胎儿及2l例足月脐带血标本取自广西医科大学第一附属医院产科2002-10／2003-02。所取的血标本均取得其家属的知情同意。②实验动物：非肥胖糖尿病／重症联合免疫缺陷小鼠12只，6-7周龄，雌性，无菌饲养于超净工作台中。 方法：采用流式细胞术，检测胎儿血的造血干／祖细胞表面标志，包括CD34,CD38，HLA-DR及CD90，同时与2l例足月儿脐血作比较。并将人胎血单个核细胞移植给6只经亚致死量照射的非肥胖糖尿病／重症联合免疫缺陷小鼠，5周后观察其植人情况，采用流式细胞术检测小鼠骨髓中人白细胞的含量，以及采用聚合酶链反应检测小鼠骨髓中的人Cart-1基因。 主要观察指标：①胎血和脐血造血干／祖细胞表面标志的表达情况。②将胎血细胞移植给非肥胖糖尿病／重症联合免疫缺陷小鼠的植入情况。结果：①人胎血中CD34^＋细胞的百分率显著高于足月脐血[（2．2588&;#177;O．7209）％，（1．5729&;#177;0．4783）％，P=0.00041，CD34^＋CD38^-细胞和CD34^＋CD90^＋细胞的百分率也均显著高于足月脐血[（1．2986&;#177;0.4706）％。（0.87l0&;#177;0.4095）％。P=0．0016；（0.9300&;#177;0.4692）％，（0.560&;#177;0.3658）％，P=0.03241。②6例胎血中的4例可顺利重建经亚致死量照射的非肥胖糖尿病／重症联合免疫缺陷小鼠的造血，移植后5周在小鼠骨髓中仍可检测到人的白细胞和人Cart-l基因。 结论：人胎儿血中有比足月脐血更高含量的造血干／祖细胞，其单个核细胞能植入非肥胖糖尿病／重症联合免疫缺陷小鼠骨髓，并重建髓、淋巴系全面造血。胎儿血有望成为多能造血干细胞来源。     

抗小鼠CD122抗体促进脐血CD34^＋细胞在NOD/SCID小鼠体内的重建

本研究旨在探讨抗小鼠CD122抗体促进人脐血CD34^＋造血干细胞在非肥胖糖尿病/重症联合免疫缺陷（NOD/SCID）小鼠体内的重建能力。对NOD/SCID小鼠进行半致死剂量γ射线照射,照射后腹腔注射200μg同型对照抗体或者抗小鼠CD122抗体,6-8 h后经尾静脉注射人脐血CD34^＋细胞或者注入PBS作为对照。处理后第2、3、4周取仅注射抗小鼠CD122抗体或同型对照抗体的小鼠外周血,动态监测小鼠NK细胞比例变化。在经过脐血CD34^＋细胞移植的小鼠中,利用流式细胞术对移植后小鼠骨髓中人CD45^＋比例以及CD45^＋细胞亚群中人CD34,CD19和CD33比例进行分析,观察抗小鼠CD122抗体对人造血干细胞在小鼠体内重建能力的影响。结果表明,用抗CD122抗体处理后2周和3周时,小鼠外周血中NK细胞比例分别为（4.6±0.6）%和（5.7±1.7）%,与注射同型对照抗体的NOD/SCID小鼠（12.2±1.4）%和（13.3±1.2）%相比下降约60%。在移植了人脐血CD34^＋细胞的小鼠中,同时用CD122抗体处理组在移植后6周和8周时小鼠骨髓中h CD45^＋比例分别为（63.0±12.2）%和（53.2±16.3）%,明显高于同型对照抗体组中h CD45^＋比例（7.7±3.6）%和（6.1±2.4）%。此外,经过抗CD122抗体处理组的小鼠移植8周后骨髓中仍能够明显的检测到人CD34^＋细胞的存在。结论：抗小鼠CD122抗体通过降低NOD/SCID小鼠的NK细胞的比例,大大促进了人脐血CD34^＋造血干细胞在免疫缺陷小鼠体内的重建能力。     

NOD/SCID小鼠模型在人脐血体外扩增后移植中的应用

造血细胞体外扩增时总细胞及祖细胞数量均倍增,但干细胞是否扩增的证据不足。动物移植模型是检测干细胞扩增的最佳实验。最近建立了一种人-非肥胖糖尿病重症联合免疫缺陷(NOD/SCID)小鼠模型,可有效评估干细胞体外扩增后体内再植力水平。本文就NOD/SCID小鼠模型的发展、影响造血细胞植入的因素及其在评估造血细胞体外扩增后体内再植力的应用进行综述。     

NOD／SCID小鼠模型在人脐血体外扩增后移植中的应用

造血细胞体外扩增时总细胞及祖细胞数量均倍增，但干细胞是否扩增的证据不足。动物移植模型是检测干细胞扩增的最佳实验。最近建立了一种人－非肥胖糖尿病重症联合免疫缺陷（NOD/SCID）小鼠模型，可有效评估干细胞体外扩增后体内再植力水平。本文就NOD／SCID小鼠模型的发展、影响造血细胞植入的因素及其在评估造血细胞体外扩增后体内再植力的应用进行综述。     

脐血造血干/祖细胞研究进展

脐血和骨髓均富含造血干/祖细胞,但二者在造血干/祖细胞的含量上存在差别。脐血中各CD34~+细胞亚群有着独特的免疫表型。脐血造血干/祖细胞能在体外扩增,扩增后的CD34~+细胞能在SCID小鼠体内重建造血。由脐血CD34~+细胞可以生成淋巴细胞和内皮细胞。脐血造血干/祖细胞移植与骨髓造血干/祖细胞移植在患者的生存率、复发率及移植免疫排斥等方面均具有可比性,有着极大的潜在价值。     

小鼠骨髓腔内输注人脐血造血干/祖细胞植活水平的观察

目的 探讨小鼠骨髓腔内输注能否增强人脐血造血干/祖细胞(HS/PC)异种移植的植活能力.方法 将不同数量(1×103、1×104、0.5×105、1×105、5×105)的人脐血CD34+细胞经尾静脉和骨髓腔途径移植入经亚致死剂量照射的NOD/SCID小鼠.于指定时间点处死小鼠,用PCR法检测人17号染色体α-微卫星特异性片段及用流式细胞术检测人源CD45+细胞,观察脐血CD34+细胞在移植小鼠左、右两侧胫骨和股骨及脾脏等部位的归巢及其长期植活能力.结果 异种移植后24h,经骨髓腔移植5×105 CD34+细胞的小鼠肝、脾、肺组织,外周血,左右两侧胫骨和股骨、骨髓细胞均表达人17号染色体α-卫星特异性片段.不同数量的人脐血CD34+细胞经骨髓腔途径移植入NOD/SCID小鼠后,8周时其植活良好(检测部位包括输注部位右侧胫骨,以及非输注部位右侧股骨、左侧胫骨、左侧股骨、脾脏及外周血).分别经尾静脉和骨髓腔两种途径输注同一来源的人脐血CD34+细胞1.0×105,8周时两组间人造血细胞植活水平分别为(44.063±20.095)%和(45.881±22.316)%,差异无统计学意义(P＜0.05);而将移植CD34+细胞数降至1.0×104时,则经骨髓腔途径输注的人脐血CD34+细胞小鼠植活水平(54.019±31.338)%显著优于尾静脉输注途径[(12.197±10.350)%,P＜0.01)];当CD34+细胞输注量降至1.0×103时,仅有骨髓腔内输注组小鼠能见到人脐血CD34+细胞植活,且植活部位通常为非输注部位骨骼.结论 小鼠骨髓腔内移植能够增强人脐血造血干/祖细胞的植活水平.     

人胸腺和脐血造血干/祖细胞联合移植对裸鼠T细胞免疫功能影响

目的:建立人胸腺和脐血造血干/祖细胞联合移植裸鼠动物模型,探讨胸腺对造血干细胞发育分化的机制.方法:实验组为人胸腺与脐血CD34+细胞联合移植.对照组只给予人脐血CD34+细胞移植,通过观察裸鼠移植胸腺CD3、HLA-DR的表达、人源CD3+细胞在小鼠脾脏的表达、裸鼠血液CD3+、CD4+、CD8+和CD4+ CD25+细胞的百分比,以及联合移植裸鼠对移植肿瘤的排斥能力,判断联合移植对裸鼠免疫功能的影响.结果:移植胸腺在裸鼠肾被膜下存活,表达CD3和HLA-DR分子;裸鼠脾脏中检测到人源CD3+T细胞呈点块状分布;裸鼠血液中CD3+、CD4+、CD8+、CD4+CD25+细胞均增高,与对照组比较差异有统计学意义(P<0.05);联合移植组能抵抗肿瘤细胞的生长,单独脐血造血干/祖细胞移植组不能抑制肿瘤生长.结论:裸鼠移植人胸腺能促进人脐血造血干/祖细胞向T细胞分化、促进免疫功能重建.     

人骨髓基质细胞促进脐血CD34+细胞的体外扩增和对NOD/SCID小鼠的植入

目的 研究人骨髓基质细胞(MSC)促进脐血CD34+细胞体外扩增及植入能力的作用.方法 分离、培养正常人的MSC作为滋养层细胞.在TPO、SCF、FL和G-CSF刺激下,比较有、无MSC滋养层细胞对扩增脐血CD34+细胞后,CD34+细胞和CFU数的增加倍数,以及植入非肥胖性糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠的能力.结果 以骨髓MSC为滋养层细胞的培养体系可以更加有效地扩增脐血CD34+细胞.体外扩增1周总细胞数(TNC)、CD34+细胞和CFU的均数分别增加111.6、19.3和58.0倍;体外扩增2周后TNC、CD34+细胞和CFU的均数分别增加532.8、41.3和563.5倍.脐血细胞输入NOD/SCID小鼠6周后,移植未扩增脐血细胞的对照组小鼠骨髓细胞中人CD45+细胞比例仅为1.2%～3.7%;移植单纯细胞因子刺激扩增组小鼠骨髓中,人CD45+细胞比例为7.6%～12.1%;以MSC为滋养层扩增的脐血细胞移植组,人CD45+细胞比例达到45.3%～59.1%.结论 以人骨髓MSC为作为饲养层细胞,不但可以更加有效扩增脐血CD34+细胞,而且可以促进脐血细胞植入NOD/SCID小鼠的能力,有潜在的临床应用价值.     

脐血造血干／祖细胞研究进展

脐血和骨髓均富含造血干／祖细胞，但二者在造血干／祖细胞的含量上存在差别。脐血中各CD34^ 细胞亚群有着独特的免疫表型。脐血造血干／祖细胞能在体外扩增，扩增后的CD34^ 细胞能在SCID小鼠体内造血，由脐血CD34^ 细胞可以生成淋巴细胞和内皮细胞。脐血造血干／祖细胞移植与骨髓造血干／祖细胞移植在患者的生存率、复发率及移植免疫排斥等方面均具有可比性，有着极大的潜在价值。     

人脐血间充质干细胞促进脐血CD34+细胞在NOD/SCID小鼠体内植入的实验研究

目的探讨人脐血间充质干细胞(MSC)联合人脐血CD34+细胞移植,能否促进CD34+细胞在NOD/SCID小鼠体内植入及加速其造血恢复.方法NOD/SCID小鼠于60Co 2.5 Gy照射后24h内由尾静脉输注胎儿脐血CD34+细胞1×105/只(低细胞量移植组)或1×106/只(高细胞量移植组),联合移植组同时输注脐血MSC 1×106/只.动态观察移植后小鼠外周血白细胞、血红蛋白和血小板恢复情况,于移植后第8周用流式细胞术检测存活小鼠骨髓中人CD45+、CD45+CD3+、CD45+CD19+和CD45+CD33+细胞的含量.结果①低细胞量移植时,联合移植组的植入率明显高于单纯移植组,分别为26.02%和16.52%(P＜0.05);高细胞量移植时,联合移植组和单纯移植组的植入率相近,分别为43.71%和39.23%(P＞0.05).②高细胞量联合移植组和单纯移植组的存活率分别为80%和70%;低细胞量联合移植组和单纯移植组的存活率分别为70%和50%.③无论高细胞量还是低细胞量组,联合移植小鼠白细胞、血红蛋白和血小板的恢复明显早于单纯移植组.④移植8周后,小鼠骨髓中人CD45+CD19+、CD45+CD33+细胞含量在低细胞量移植时,联合移植组高于单纯移植组;但在高细胞量移植时,两组之间差异无统计学意义.CD45+CD41a+细胞的含量无论在低细胞量和高细胞量移植时,联合移植组均高于单纯移植组.各组小鼠骨髓中CD45+CD3+细胞的含量均较少,且各组之间差异无统计学意义.结论①低细胞量移植时,人脐血MSC联合移植可提高人脐血CD34+细胞在小鼠体内的植入率.②人脐血MSC与人脐血CD34+细胞联合移植,加速NOD/SCID小鼠各系造血恢复,提高移植小鼠存活率.③MSC联合移植可促进人脐血CD34+细胞在NOD/SCID小鼠体内向粒系、B淋巴系和巨核系定向分化.     

不同来源人造血干/祖细胞在NOD/SCID小鼠体内归巢能力的差异

目的 比较不同来源的人造血干／祖细胞在NOD／SCID小鼠体内归巢能力的差异性，并探讨其体内归巢能力与膜表面归巢相关分子CXCR4表达水平的相关性。方法 采用流式细胞术（FACS）检测新鲜脐血、冻存脐血、动员后外周血（mPB）及骨髓来源的CD34^＋细胞表面CXCR4表达水平；将荧光染料CFSE标记的人CD34^＋细胞移植人接受照射的NOD／SCID小鼠，移植后20小时检测已归巢于NOD／SCID小鼠骨髓及脾脏中不同来源的人CD34^＋细胞，计算其相应的骨髓及脾脏归巢效率；并将小鼠股骨制成组织切片，荧光显微镜下观察人CD34^＋细胞在小鼠骨髓腔内的分布。结果 新鲜脐血、冻存脐血、mPB和骨髓CD34^＋细胞膜表面CXCR4表达阳性率分别为（49.52±1．12）％。（46．12±2．29）％，（48．50±2．48）％和（65．39±1．27）％，CD34^＋细胞在NOD／SCID小鼠骨髓的归巢效率分别为（3．00±0．44）％，（2．84±0．46）％，（4．06±0．70）％及（5．76±0．52）％；在脾脏的归巢率分别为（1．88±0．12）％，（1．80±0．15）％，（1．90±0．22）％，（2．16±0．34）％。归巢的CD34^＋细胞主要分布于小鼠股骨的骨内膜区域。结论 脐血CD34^＋细胞膜表面CXCR4水平低于mPB和骨髓。经冻存复苏后脐血CD34^＋细胞膜表面CXCR4水平略有下调。脐血CD34^＋细胞在照射NOD／SCID小鼠的骨髓归巢效率低于mPB和骨髓。     

NOD/SCID小鼠脐血单个核细胞骨髓腔内移植的实验研究

目的观察骨髓腔内移植(iBM)人脐血单个核细胞(MNC)对小鼠造血重建和免疫功能恢复的作用。方法NOD/SCID小鼠经137Cs全身照射后,在4h内无菌条件下局部麻醉后从尾静脉或骨髓腔内输注分离脐血MNC。受体小鼠随机分为5组:①对照组:骨髓腔内输注培养液;②阳性对照组(iTV):尾静脉输注脐血MNC3×107/只;③实验Ⅰ组(iBM1):骨髓腔内输注脐血MNC3×106/只;④实验Ⅱ组(iBM2):骨髓腔内输注脐血MNC1×107/只;⑤实验Ⅲ组(iBM3):骨髓腔内输注脐血MNC3×107/只。对照组4只小鼠,实验Ⅱ组7只小鼠,其余每组5只。24h后观察iBM22只小鼠未移植侧胫骨骨髓腔脐血细胞的迁移分布,动态观察移植后小鼠的存活和造血重建情况,7～8周后处死各组小鼠,检测骨髓细胞表面CD分子表达、碳青花(DilCM)染料示踪研究和脐血βactin的DNA标记。结果①照射后骨髓腔内输注脐血MNC,24h后在未输注脐血MNC的一侧胫骨骨髓细胞膜有DilCM标记;②7～8周后小鼠存活14只,其中对照组存活1只,iTV组、iBM1组各存活2只,iBM2组存活4只,iBM3组存活5只;③外周血常规检查结果显示iBM组白细胞的恢复速度比iTV组和对照组快而且稳定;④移植后存活小鼠骨髓细胞表面CD45标记、DilCM染料示踪研究和βactin均显示人源的标记。结论经iBM途径移植脐血MNC至NOD/SCID小鼠骨髓可以重     

间充质干细胞与人脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响

目的 观察间充质干细胞(MSC)与不同比例脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响,明确MSC与脐血CD34+细胞共移植的最适数量.方法 给60Coγ射线照射的雌性NOD/SCID小鼠共移植人MSC和不同比例的脐血CD34+细胞,观察共移植后42 d内小鼠外周血白细胞和血小板变化,并于移植后42 d处死小鼠,用流式细胞术检测外周血、骨髓和脾脏人源细胞含量.结果 与单纯脐血CD34+细胞移植相比较:①脐血CD34+细胞与1、5和10倍数量的MSC共移植时,可明显减轻外周血白细胞和皿小板的下降幅度(P<0.01),提前1周使白细胞和血小板恢复至正常水平(P<0.05),三组间差异无统计学意义(P>0.05);②MSC与不同比例的脐血CD34+细胞共移植均可明显提高外周血、骨髓和脾脏造血细胞植入率.比例为10:1时,外周血、骨髓和脾脏中的人源细胞(huCD45+细胞)含量分别增加了(2.8±0.6)倍、(3.5±0.9)倍和(5.2±0.6)倍,增加倍数差异均有统计学意义(P<0.01),达到了最佳的植入效果.结论 脐血CD34+细胞与10倍数量的MSC共移植可达到最佳的促进造血重建作用.     

Multiorgan engraftment and multilineage differentiation by human fetal bone marrow Flk1+/CD31-/CD34- Progenitors

We previously reported that Flk1(+)/CD31(-)/CD34(-) cells isolated from human fetal bone marrow can differentiate at the single cell level into endothelial and hematopoietic cells in vitro. Here we report that within this cell population reside cells that can differentiate into the epithelium of liver, lung, gut, as well as the cells of both hematopoietic and endothelial system after primary or secondary transplantation into irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Hence, Flk1(+)/CD31(-)/CD34(-) cells possess remarkable differentiation potential and may thereby provide an alternative to hematopoietic stem cells for transplantation. In addition, our results show this stem cell population effectively accelerated wound healing in NOD/SCID mice and thus holds therapeutic promise for treatment of genetic disorders, organ dysfunction, and tissue repair in humans.     

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.     

Repopulating activities of human cord blood cells separated by a stem cell collection filter in NOD/SCID mice: a comparative study of filter method and HES method

BACKGROUND: Volume reduction and removal of RBCs are essential for cost-efficient cord blood (CB) banking. It has previously been shown that a newly developed device, a stem cell-collection filter (SCCF), can reduce the CB volume and remove RBCs efficiently, giving high recovery rates for CD34+ cells, colony-forming cells, and long-term culture-initiating cells with short operation time. The aim of this study was to compare the quality of CB cells separated by SCCF and HES by analyzing repopulation in NOD/SCID mice. STUDY DESIGN AND METHODS: A total of 1 x 10(6) or 5 x 10(6) nucleated cells derived from SCCF- or HES-separated, cryopreserved, thawed, and washed CB were transplanted into NOD/SCID mice. Eight weeks after transplantation, bone marrow cells of the recipient mice were examined by flow cytometry and hematopoietic progenitor assay for the engraftment of human cells. RESULTS: Mice given human CB cells, separated by SCCF, showed degrees of engraftment similar to those in mice given HES-separated CB cells. There was no significant difference in the lymphohematopoietic reconstitution pattern in the two groups of mice. CONCLUSION: SCCF processing does not appear to reduce the number of repopulating cells in NOD/SCID mice or alter the number of HPCs. It is now shown that these cells can be captured by SCCF and removed, and that they will engraft.     

Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice

Increasing use of purified or cultured human hematopoietic cells as transplants has revealed an urgent need for better methods to predict the speed and durability of their engraftment potential. We now show that NOD/SCID-beta2 microglobulin-null (NOD/SCID-beta2m-/-) mice are sequentially engrafted by two distinct and previously unrecognized populations of transplantable human short-term repopulating hematopoietic cells (STRCs), neither of which efficiently engraft NOD/SCID mice. One is predominantly CD34+CD38+ and is myeloid-restricted; the other is predominantly CD34+CD38- and has broader lymphomyeloid differentiation potential. In contrast, the long-term repopulating human cells that generate lymphoid and myeloid progeny in NOD/SCID mice engraft and self-renew in NOD/SCID-beta2m-/- mice equally efficiently. In short-term expansion cultures of adult bone marrow cells, myeloid-restricted STRCs were preferentially amplified (greater than tenfold) and, interestingly, both types of STRC were found to be selectively elevated in mobilized peripheral blood harvests. These results suggest an enhanced sensitivity of STRCs to natural killer cell-mediated rejection. They also provide new in vivo assays for different types of human STRC that may help to predict the engraftment potential of clinical transplants and facilitate future investigation of early stages of human hematopoietic stem cell differentiation.